Hairpin-like secondary structures in DNA and its transcripts are thought to play a crucial role in gene regulation, serving as potential recognition and interaction sites for other macromolecules in the cell. In spite of much evidence for their biological significance in exact structural and/or sequence requirements for a biologically active hairpin remains unknown. It appears, however, that the stem length of a hairpin is a crucial determinant for biological activity. I propose herein to investigate the correlation between the size and stability (sequence) of a hairpin and its biological acitivity. The genetically and biochemically well-defined system of bacteriophage PhiX174 will be employed. Oligonucleotides of defined invert repeat sequence will be inserted into the non-essential J-F intercistronic region of the phage genome by a newly devised scheme of site specific mutagenesis and biochemcial selection of mutant genomes. A series of phage mutants will be constructed which have the potential to form perfectly base paired hairpins of increasing size in a region of their genome which is known to influence a variety of functions. Formation of these structures in supercoiled RF (replicative form) DNA and its consequence for neighboring secondary structrures will be analyzed with single strand specific nucleases as probes for hairpin loops. Biological studies will be performed to analyze the effect of hairpin size and stability on phage replication and the synthesis of phage proteins. Supercoiled and linear RF DNA, as well as fragments thereof, will be used as templates for in vitro transcription assays to specifically test the effect of hairpin conformation on transcription termination. The ability of ribosomes to bind to DNA will be used to measure the effect of secondary structures on translational initiation. Collaborating investigators will study the phage mutants with regard to in vivo RNA synthesis and phage eclipse kinetics. These studies are aimed to further our knowledge of how nucleic acid secondary structure is involved in the regulation of gene expression. Exploring these mechanisms is crucial to our understanding of those molecular events that underly viral replication, cellular differentiation and its reversal during malignant transformation.